Avicenna Journal of Phytomedicine Received: Feb 11, 2013; Accepted: Jun 19, 2013 Epub ahead of print

Short communication

Comparison between effects of different doses of Melissa officinalis and atorvastatin on the activity of liver enzymes in hypercholesterolemia rats

Ali Zarei 1, Saeed Changizi Ashtiyani *2, Soheila Taheri 3, Fateme Rasekh 4

Abstract Objectives: Liver is one of the most sensitive tissues to oxidant damage. Hence, the present study was conducted to compare the effects of Melissa officinalis (MO) extract and Atorvastatin on the activity of liver enzymes in rats. Materials and Methods: In this experimental study, 60 male Wistar rats were selected and allocated to six groups (n=10). The control group received a normal diet, sham group received a fatty diet while other groups received a fatty diet and the alcoholic extract of MO, at minimum (25 mg/kg), moderate (50 mg/kg), and maximum (75 mg/kg) doses (i.p.). The last group received Atorvastatin (10 mg/kg) through gavage with a fatty diet over a 21-day period. At the end of this 21-day period, blood samples were drawn and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), activity of liver enzymes as well as cholesterol in the samples were measured. Results: The obtained results showed that the activity of liver enzymes in the treatment groups receiving MO extract and the group receiving Atorvastatin decreased significantly. MO extract reduced the level of liver enzymes. Conclusion: Therefore, further studies for obtaining a better understanding of the mechanism of effect of MO for treating liver diseases are recommended.

Keywords: Atorvastatin, Hypercholesterolemia , Melissa officinalis, liver Enzyme, Rat

1- Department of Biology, Unit of Science Research, of Damghan, Damghan, I.R. 2- Department of Physiology, Arak University of Medical Sciences, Arak, I. R. Iran 3- Deputy of Research, Arak University of Medical Sciences, Arak, I. R. Iran 4- Department of Plant Biology, Payame-Noor University, , I. R. Iran * Corresponding Author: Tel: +9808614173638; Fax: +9808614173639 E-mail: [email protected]

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Introduction compounds, such as quercetin , gallic acid, as The study of compounds with herbal well as flavonoids, aldehyde, and tannin origin is an interesting branch of medicine compounds (Pereira et al., 2009). Due to its (Akhondzadeh et al., 2003). Currently, a medicinal properties, M. officinalis has been great deal of attention is paid to the used in other studies for examining its effects consumption of fruits and vegetables for their on Alzheimer, memory, learning, and protective effects against cancer, depression. However, to date no study has cardiovascular diseases, and liver diseases been done to compare the effects of M. (Perez-Carreon et al., 2002; Pyo et al., 2004). officinalis extract and Atorvastatin on the This is due to the presence of antioxidant amount of activity of liver enzymes in compounds including vitamin C, Vitamin B, hypercholesterolemic rats. Therefore, the Carotenoids , lycopens, and flavonoids that present study was done to compare the prevent damage caused by free radicals effects of M. officinalis extract and (Mitra et al., 1998; Sun, 2002; Yang et al., Atorvastatin on the activity of liver enzymes 2001). in hypercholesterolemic rats. Melissa officinalis (M. officinalis) or Lemon balm belonging to the Lamiaceae family is a medicinal plant native to East Materials and Methods Mediterranean region and West Asia. In Iran, This experimental study was done on 60 this plant is known as Badranjbooye and male Wistar rats supplied from the Animal grows largely in Tehran, Golestan, Breeding Center of University of Azarbayjan, Lorestan, and (Emamghorishi and Talebianpour, 2009; Medical Sciences. The rats were kept in Zargari, 1991). Since ancient times, this herb standard conditions of temperature and light has been used for its antispasmodic, sedative, ad libitum . Animal care and handling was hypnotic, nutritional, anti-flatulent, and performed according to the guidelines and perspiration properties (Emamghorishi and ethical codes set by the Iranian Ministry of Talebianpour, 2009; Blumenthal et al., 2000). Health and Medical Education for lab M. officinalis also enhances memory and animals. Before launching the project, all of relieves stress (Akhondzadeh et al., 2003; the rats were weighed to make sure that they Schulz et al., 1998) and it is applied to the were within the same range of weight (170±5 treatment of sore throat, herpes, and g). headache (Emamghorishi and Talebianpour, The rats were randomly assorted into 6 2009; Vessal et al., 1996). groups of 10 each and received treatment for In Iran, it is used for treating bad-temper, 21 days in the following way: anxiety, and nervousness in young girls and 1) Control group: The rats did not receive women (Emamghorishi and Talebianpour, any vehicle or drugs and had a normal diet. 2009; Zargari, 1991; Shafie-Zadeh, 2002; 2) Injection sham group: Imami et al., 2003). M. officinalis can be Hypercholesterolemic rats (cholesterol 2% effective in the treatment of fever, was added to their food to render them indigestion, insomnia, and epilepsy as well hypercholesterolemic) were daily (De Carvalho et al., 2011). One of the main administered 0.2 ml of normal saline (i.p.). properties of M. officinalis is its antioxidant 3) Treatment group 1: Hypercholesterolemic property (Pereira et al., 2009) which is due to rats were administered 25 mg/kg (minimum the presence of special compounds in it. M. dose) of the alcoholic extract of M. officinalis officinalis extract has polyphenolic (i.p.).

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4) Treatment group 2: Hypercholesterolemic temperature for 20 min, and centrifuged for rats were administered 50 mg/kg (moderate 20 min at 2000 rpm. The supernatant was dose) of the alcoholic extract of M. officinalis transferred into test tubes for lipid analysis. (i.p.). 5) Treatment group 3: Hypercholesterolemic Extraction method rats were administered 75 mg/kg (maximum For preparation of the alcoholic extract dose) of the alcoholic extract of M. officinalis of M. officinalis (Figure 1) standard methods (i.p.). of extraction were utilized. After providing 6) Atorvastain group: In this group, 10 mg/kg M. officinalis plant, it was cleaned, dried, of atorvastain in the form of oral emulsion powdered, and eventually poured into a was administered to hypercholesterolemic capped glass container and was mixed with rats through gavage. ethanol alcohol 96%. The mixture was The experiment was carried out over a 21- allowed to mix well for 72 hours. After that, day period and the injections were done it was first filtered and then centrifuged. The through gavage everyday at 9 a.m. Injections resulting mixture was placed in a hot bath to were made intraperitoneally using insulin allow its alcohol evaporate completely. After syringe and Atorvastatin (10 mg/kg) (Shafa this stage, due to the presence of water, the Pharmaceuticals Co., Iran) was administered extract was still in a liquid state and for in the form of an oral emulsion. complete evaporation of water, it was kept at ° After this period, mild anesthesia using 40 C and then placed adjacent to sodium ether was done for obtaining blood samples chloride. from the rats heart to examine activities (IU/L) of serum liver enzymes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), as well as the amount of cholesterol in the samples. After centrifuging the blood samples at 3500 rounds per minute, serum samples were isolated and transferred to the lab for measuring the intended factors. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined colorimetrically according to the method of Reitman and Frankel (Retiman Figure 1. Melissa officinalis leaves. and Frankel, 1957). Alkaline phosphatase (ALP) was assayed according to the method Preparation of high cholesterol 2% food described by Belfield and Goldberg (Belfield For preparing high cholesterol 2% food, and Goldberg, 1971). 20 g of pure cholesterol powder (Fluka Chemika) was solved in 5 ml of heated olive Analysis of cholesterol in plasma oil and was mixed with 1 kg of the rats food. Plasma cholesterol level was determined All of the treatment groups received a fatty by enzymatic colorimetric methods, using diet during the experiment. commercial kits from Pars Azmoon (Tehran, Iran) (Bhandari et al., 1998). Briefly, blood Data analysis samples were transferred directly into All obtained values are expressed as centrifuge tubes, allowed to clot at room mean±Standard Error (SE) and data analyses

Epub ahead of print 3 Zarei et al. were done using SPSS version 11.5 statistical control group, whereas its amounts in all of software. The mean values were compared the treatment groups receiving M. officinalis using one-way ANOVA analysis followed by extract as well as the Atorvastatin group had Tukey’s post-hoc test. The level of significantly decreased (p=0.001). significance was set at 0.05. ALP value in the sham group was significantly increased in comparison with the control group, whereas changes in all of Results the treatment groups receiving the extract The comparison between the results of and the group receiving Atorvastatin statistical analysis on the effects of M. presented significant decreases compared officinalis extract and Atorvastatin on the with the sham group (p=0.00). Moreover, all activity level of liver enzymes and treatment groups showed significant cholesterol in hypercholesterolemic rats increases in ALP values compared with the (Table1) indicated that the amount of ALT Atorvastatin group (p=0.00). However, the and cholesterol in the injection sham group treatment groups did not present significant receiving a fatty diet significantly increased changes compared with one another. in comparison with the control group, Moreover, plasma concentrations of whereas changes in all of the treatment cholesterol in groups receiving different groups receiving the extract and the group doses of the alcoholic extract of M. receiving Atorvastatin presented significant officinalis showed significant reductions in decreases compared with the injection sham comparison with the injection sham group group (p=0.00). However, none of the (Table 1). In this study, no significant treatment groups presented significant differences in activity of liver enzymes changes compared with one another and the between moderate dose of M. officinalis Atorvastatin group. extract with its minimum and also maximum In terms of AST, changes in the sham doses were found (Table 1). group were significantly higher than the

Table 1. The comparison between effects of different doses of Melissa officinalis (MO) versus Atorvastatin on activities (IU/L) of serum liver enzymes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and cholesterol in hypercholesterolemia rats.

Group MO MO MO Atorvastatin Control Sham Minimum dose Moderate dose Maximum dose (10 mg/kg) Parameters (25 mg/kg) (50 mg/kg) (75 mg/kg)

ALT (IU/L) 55.87±3.7 80.37±6.7 58.75±2 62.96±2.2 68.12±1.4 65.28±2.4 * † † † †

AST (IU/L) 234.8±15.4 292±32.6 183±15.9 205±21.2 191±10.9 177.43±4.8 * † † † †

ALP (IU/L) 1017.3±71.8 2400±44.5 1485±63.9 1520±95.9 1454±10.5 673.6±12.1 * †# †# †# †

Cholesterol 85±1 97.3±3.1 86.0±5.4 86±4.0 76.12±5.1 80.62±2 (mg/dl) * † † † †

† Comparison with the sham group; # Comparison with the Atorvastatin group ; * Comparison with the control group .

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Discussion quercine compounds which can inhibit lipid Statistical analysis of the results showed peroxidation (Pereira et al., 2009; -Saija et al. that M. officinalis extract and Atorvastatin 1995), its extract stabilizes cell membrane equally decreased liver enzymes. M. and prevents the oxidation of membrane officinalis possesses strong antioxidant lipids. Hence, in the present study, decrease properties and it has phenolic compounds in activity of liver enzymes in the treatment (Pereira et al., 2009). Polyphenolic groups receiving M. officinalis is justifiable. compounds are among the most important Moreover, the presence of non-enzymatic anti-oxidants (Pérez-Carreón et al., 2002; antioxidants, including vitamin E, beta Pyo et al., 2004; Toit et al., 2001). These carotene, and vitamin C in this herb justifies compounds, especially flavonoids, have its ability to inhibit damage due to free protective effects on liver damage induced by radicals. The involved enzymes are free radicals and liver toxins (Ahmad et al., superoxide dismutase, catalase, and 2002; Germano et al., 1999; Yoshikava et al., glutathione peroxidase. When the anti- 2003). oxidant defense system is lost in the body, M. officinalis is applied to the treatment of increase in the formation of free radicals can Alzheimer. Research has shown that this herb cause cellular oxidative stress. Lipid can calm the patients in their behavior, peroxidation and oxidative stress severely improve their learning, and enhance their increase in patients with diabetes and short-term memory (Akhondzadeh et al., hyperlipidemia (Kelly and Husband, 2003; 2003). There was also considerable variation Nazari et al., 2005). in cholinoreceptor interactions between Polyphenolic compounds and flavonoids different accessions of a M. officinalis can also revive the cells against glutathione species. M. officinalis extracts showed some depletion and protect them by increasing the nicotinic and muscarinic activity capacity of anti-oxidant enzymes (Akhondzadeh et al., 2003; Wake et al., (glutathione, glutathione reductase, 2000). Moreover, it has also been shown that glutathione peroxidase, and catalase) (Sanz et consumption of M. officinalis can be al., 1998; Al–Qarawi et al., 2002). uplifting and brings about euphoria Free radicals damage cell membrane, such (Akhondzadeh et al., 2003; Kennedy et al., as in hepatocytes. With destruction of 2002). The findings of the present study hepatocytes membrane, the activity of liver indicated that similar to Atorvastatin, M. enzymes increases which results in the officinalis extract had an attenuating effect expression of these enzymes, which are on activity of liver enzymes in the treatment normally in cytosol of cells. Increase in the groups. Polyphenolic compounds due to their activity of these enzymes explains the type antioxidant properties can neutralize free and degree of liver damage (Shariati and radicals in the environment and prevent their Zarei, 2006). Considering the properties of destructive effects (Perez-Carreon et al., this herb which decrease the production of 2002; Sun, 2000; Schulz et al., 2004). free radicals and its anti-oxidant and anti- Active free radicals, such as superoxide inflammatory properties, reduction in the anions and hydroxyls, are capable of level of activity of liver enzymes in the removing hydrogen atoms from the treatment groups receiving M. officinalis peripheral chains of saturated fatty acids in extract is predictable. In the present study, it biologic membranes and result in lipid was shown that treatment with M. officinalis peroxidation (Kelly and Husband, 2003; extract can protect liver cells which are Nazari et al., 2005). Since M. officinalis has probably due to the presence of flavonoids.

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Research has shown that M. officinalis secretion of VLDL containing triacylglycerol possesses anti-inflammatory properties. and bad cholesterol by liver can decrease There is a direct relationship between the plasma cholesterol. amount of lipids and leptin. Furthermore, The results of a study by Lee and with increase in the amount of lipids and coworkers showed that in vivo and in vitro their deposition in the liver, the level of treatments of Ob-X (a mixture of three herbs, activity of liver enzymes increases. Increase Morus alba , Melissa officinalis , and in the amount of lipids results in an increase Artemisia capillaries ) regulate serum lipid in the level of leptin (Saeb et al., 2010; profiles, adipose tissue mass, and body Jelodar and Nazifi habibabad, 1997, Bush, weight gain by inducing the mRNA 1992; Tohidi, 2007). Leptin secretion expression of peroxisome proliferator- increases with stimulation of inflammation activated receptor (PPAR) target genes and it increases hormonal and cellular responsible for fatty acid oxidation in high- immune responses (Gainsford et al., 1996). fat diet-fed obese mice. Thus, hepatic PPAR Moreover, adipocytes release various protein seems to be a crucial component for Ob-X signals that include several cytokines such as regulated energy metabolism and Ob-X may TNF-α, IL-6, and absorbing proteins be very useful for treating obese patients with (Hersoug and Linneberg, 2007). By hypercholesterolemia and activating the secretion of TNF-α and IL-6 hypertriglyceridemia. from mononucleus cells in cytokines, leptin In general, methods of reducing can cause inflammation (Gatreh-Samani et cholesterol include decreasing its excretion al., 2011). TNF-α increase is probably and inhibiting its synthesis and absorption. followed by liver necrosis which leads to an Studies done on M. officinalis also indicate increase in activity of liver enzymes (Shariati that it contains phenolic alkaloids that are and Zarei, 2006). among the materials which can inhibit Noticing the anti-oxidant and anti- cholesterol synthesis (Pereira et al., 2009; inflammatory properties of Melissa Ashtiyani et al., 2011). The results of this officinalis, reduction in the level of activity study is in agreement with Bolkent S and of liver enzymes in the treatment groups was coworkers that showed that the predictable. Alkaline phosphatase is a administration of M. officinalis L. extract transpeptidase which increases in liver and reduced total cholesterol, total lipid, ALT, bone diseases. Research has shown that the AST, and ALP levels in serum as well as phenolic compound present in medicinal lipid peroxidation (LPO) levels in liver plants can prevent the toxic effects of drugs tissue. Moreover, they showed increased on liver and cause the release of serum ALT glutathione levels in the tissue (Bolkent et al, into blood. Studies have demonstrated that 2005). As a result, it was suggested that M. hyperlipidemia levels, glycerides, and officinalis extract exerted a hypolipidemic cholesterol in groups under treatment with effect similar to Atorvastatin and showed a fatty diets increase (Pourjafar et al., 2010). protective effect on the liver of Atorvastatin is one of the chemical drugs hyperlipidemic rats. that are used for treating hyperlipidemia. Previous studies and studies done together Drugs reducing blood fat, such as lovastatin with this one show that M. officinalis extract and Atorvastatin, by inhibiting the channel of by decreasing AST, ALP, and ALT as well hydroxymethylglutaryl-coenzyme, a as cholesterol is effective in improving the reductase synthesis, and decreasing the function of lever and treating liver diseases.

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